Small-angle X-ray scattering study of the interaction between lysine transfer RNA ligase from yeast and transfer RNA

J. Mol. Biol. (1975) 99, 383-400

Small-angle X-ray Scattering Study of the Interaction between Lysine Transfer RNA Ligase from Yeast and Transfer RNA RAo~

0STEaBF~RO, B e SZSBERO, LARS R ~ O Am) ULV LAO~R~v~ST

Department of Medical Biochemistry, Univeraity of qSteborg G~teborg, Sweden (Received 3 ATril 1975) The main complex formed in solution between lysine:tRNA ligase from yeast and tRNA is composed of two enzyme molecules and one molecule of t R N A as shown by an equilibrium analysis of small angle X-ray scattering data recorded at 21°C in the p H range 6.6 to 7.4. The stability of the complex with both cognate and non-cognate t R N A decreases with increasing Mg 2+ concentration. The two types of complexes differ, however, in as much as the stability of the non-cognate complex is independent o f p H while the stability of the cognate complex decreases with increasing pH. Analysis of the small-angle X-ray scattering from the complex between enzyme and cognate tRNA gave a radius of gyration of 57.4 A, a molecular weight of 236,000 and a volume of 600,000 A a, in agreement with the theoretical values calculated for a complex containing two enzyme molecules and one tRNA (molecular weight 252,600 and volume 631,000 As). A comparison of the experimental data with theoretical scattering curves computed for different triaxial bodies suggests that the two ellipsoid protein molecules interact with their longest semi-axes at right angles to each other, and that within the angle formed, partly engulfed by the protein ellipsoids, is the supposedly L-shaped tRNA molecule. 1. I n t r o d u c t i o n The elucidation of the structural basis for the specific interactions between a protein and a nucleic acid is a m a j o r objective in molecular biology. The recognition b y a t R N A ligase of its t R N A substrate is a good example of this general problem and consequently has received much attention. Nevertheless, we are still far from a full understanding of the mechanism involved in the recognition procedure, a n d it is obvious t h a t this will eventually require a thorough knowledge of the structure of b o t h enzyme and t R N A and the e n z y m e - t R N A complex. While the three.dimensional structure of one t R N A has now been determined (Suddath d al., 1974; Robertus al., 1974) and X - r a y analyses of several ligases~ are presently under w a y ( R y m o et al., 1970; Waller et al., 1971; Chirikjian d al., 1972; Reid e~ a/., 1973), it has not been possible so far to obtain the l i g a s e - t R N A complex in crystalline form. The prospects Abbreviations used: tRNA TM, transfer ribonucleic acid specific for lysine; tRNA TM,transfer ribonueleic acid specific for valine. The word ligase is used in this paper to denote an amino acid:~RNA ligase. O~her notations are: A, lysine:tR~A ligase; B, tRNA; A the total concentration of the ligase; B, the total concentration of tRNA; p, q, number of A and B molecules bound in ApBq (capital P and Q are used when one single ApB¢ complex is assumed); ~p~, equilibrium constant at a constant pH defined by equation (1); I, slit "smeared" absolute intensity.

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of a single crystal analysis of such a complex are therefore very uncertain and, consequently, it seemed of considerable interest to explore other methods which could give structural information about the interaction between t R N A and ligase. This communication reports the composition and molecular parameters of a complex between l y s i n e : t R N A ligase from yeast and different t R N A s as revealed b y small-angle X - r a y scattering. The main complex formed in solution is composed of two enzyme molecules and one molecule of t R N A . The stability of the complex, both with cognate and non-cognate t R N A , decreases with increasing Mg 2+ concentration but the two complexes differ in the sense that the stability of the non-cognate complex is independent of p H while that of the cognate complex decreases with increasing

2. M a t e r i a l s a n d M e t h o d s (a) Preparation of enzyme and t R N A Lysine:tRNA ligase from ~accharomyces c~rev~iae C836 was prepared as described previously (Rymo e$ al., 1970). Protein concentrations were calculated from protein nitrogen (Rymo e$ al., 1972), which was analysed either by a micro-Kjeldahl procedure (Strid, 1961) or by a micro-Dumas method (Kirsten, 1971). The concentration of tRNA was either calculated from the nitrogen content of the tRNA solution or else from its optical density. In this case the unit used (A26o) was defined as the amount of material which, when dissolved in 1.0 ml, gave an absorbance of 1.0 at 260 nm with a light-path of 1-0 cm. When the molar concentration of tRNA was calculated from its optical density, 1-0 A~6o unit was taken to equal 1.8 umol of tRNA. Highly purified tRNA T M and tRNA TM were prepared from crude yeast tRNA obtained from Bochringer and Soehne, Mannheim, Germany, using the method described by Gillam e$ al. (1968). The product obtained in the final purification step had a ratio of esterified amino acid to A28o that was close to the value expected for pure tRNA T M and tRNA TM. After removal of the esterifying e.m~no acid the preparations gave 1.4 to 1.8 nmol of amino acid per Aa60 in the standard assay (Rymo e$ a/., 1970). (b) X-ray m ~ r e ' m e ~ The X-ray small-angle scattering data were recorded with a camera as described by Kratky & Skala (1958). I t was equipped with an electronically programmed step scanning device (Leopold, 1968). Due to the X-ray sensitivity of the l~gase, a special flow cuvette was used which ensured that a fresh solution was introduced every 30 m~u during the measurements ((Ssterberg et al., 1973). Monochromatization was achieved by using a nickel filter and a pulse height discrhninator in conjunction with a proportional counter. All measurements were made at a temperature of 21.0°C. Scattering curves used only for equilibrium calculations were recorded up to a scattering angle, 20, of 0.010 radians, while for the determination of molecular parameters, curves to an angle of 0.059 radian were measured. For every point on the curve, 4 × 10~ pulses were recorded. The scattering recorded from the buffer solution was subtracted from each scattering curve. The absolute scattered intensities were obtained by using a Lupolen standard sample (Kratky e~ ~., 1966). The Lupolen sample had been calibrated previously at the Graz Institut fGr Physikalische Chemic. (c) Equilibrium data The equ~ibria in the present systems may be described by the general reaction pA ~- qB ~ ApBq,

(1)

and the equilibrium constant ~ . Here, A stands for the lysine:tRNA ligase and B for tRNA. The standard method for measuring complex formation between ligase and tRNA was as follows. Two series of solutions, one for tRNA TM and one, as a control, for tRNA TM,

X - R A Y S C A T T E R I N G OF t R N A L I G A S E C O M P L E X

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were prepared with different concentrations of t R N A (2 to 100 ~ ) and a constau~ concentration of ligase ( ~ 2 5 ~M or ~ 5 0 ~ ) . The samples of llgase and tRNA, u~ed in each series, had previously been dialysed at q-4°C against an 0.1 ~-phosphate buffer, which in some experiments also contained Mg u +. The same phosphate buffer solution was also used for all dilutions of the samples so that each series of solutions had the same Mg u+ concentration and the same pH. After dialysis the samples were kept at -- 80°C until studied. The small-angle X-ray scattering was recorded for each sample, and the absolute scattered intensities were obtained by using a Lupolen standard sample (Kratky e~ al., 1966). X-ray scattering from the dialysis buffer was used to correct for the background scattering. For each series of ligase and t R N A solutions, the scattering curve of a sample of native ligase was recorded separately. The concentration of this ligase solution was the same as t h a t of the other solutions in this particular series. The X-ray scattering of a sample of tRNA, usually at a concentration of 1 to 2 mg/ml, was also recorded separately under the same conditions. A standard curve of absolute intensity versus tRNA concentration (mg/ml) at a certain constant angle had previously been recorded for tRNA concentrations varying from 0.5 to 5 mg/ml. I n this range it almost followed a straight line. I t should be noted that in the concentration ranges studied, the data indicated that both the t R N A and the ligase exist only in monomerie form. D a t a were recorded for the p H range, 6-6 to 7-5, both without Mg a + and in the presence of I m~-Mg 2+ ions. Additional studies of the effect of different Mg 2 + concentrations were made at p H 6-8. The p H of each buffer was determined to within 0.02 p H unit (on the relative scale to within 0.005 p H unit), using a Radiometer PHM4 p H meter and a potassium phthalate standard buffer. 3. R e s u l t s (a)

Ano~ysi8 of the ~uiliSr~um data

An example of the intensity d a t a used for the analysis of the equilibria in the present system is shown in Figure 1. F r o m such absolute intensity data, cut at one or sometimes two constant angles (usually 2.9 and/or 4.9 mradians), the composition of the main complex was determined using the method described in the Appendix. This means t h a t one m u s t first calculate the difference intensity, A1, which is a function of the complexes formed and not of the free molecules. Then, b y plotting A I / A versus the total t R N A concentration (B), the m a x i m u m value of ALIA, (AXY/A)max,is obtained (A is the t o t a l concentration of the enzyme) so t h a t the normalized difference intensity, (AT-/A)/(AI[A)max, can be calculated. B y comparing the experimental data, plotted as (AliA)/(AliA)max ver~8 B, with a set of curves generated for different assumed species and different equilibrium constants, the composition of the main species and the equilibrium constant for its formation can be determined. The analysis of the d a t a indicated t h a t a species of the t y p e A2B is formed, i.e. a complex containing two enzyme molecules and one t R N A . This was true b o t h for the cognate complex formed with t R N A T M a n d the non-cognate with t R N A TM. I t was possible to exclude the formation of other species, such as the ApB o species having Q ~ P (for instance ABo species with Q = 1,2 . . . . ), since, even for v e r y high fllQ values (Q ~ 1), the d a t a generated for such complexes initially follow a curve t h a t is to the right of our measured d a t a (see Appendix, Fig. A1). On the other hand, although a n A2B species gave the best fit, equih'brium d a t a for a single value of A do not exclude a composition of the t y p e A3B or A4B (Appendix, Fig. A1) for the m a i n species. However, additional support for the AaB species is obtained b y considering the space between two sets of d a t a having two different values of A ( ~ g . 2). Since B ~ ~aPbOfl~,o,wefind for constant (AI/A)](AJ/[A)max---- Z = 0"5: t~ (aA[SB)z= P/(QZ) ~ 4. This gives P[Q - - 2. The values of this derivative for a n AsB species 2O

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A=44-3/~M

I I

153 76

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Radians x I0 3

Radians x I0 z

FIG. 1. Absolute intensity data used for analysing the complex formation between lysine :tRNA ligase and tRNA T M from yeast. The vertical, dashed lines indicate the two constant angles for which the 4 [ data were calculated. A is the total concentration of lysine :tRNA ligase and B is the total concentration of tRI~A''y'. would be 6 and for a n A4B species 8. ~xrthermore, a complex having three or more ligase molecules does not agree with the molecular parameters obtained (cf. Table 1). Therefore, tA,Irlng both equilibrium d a t a and molecular parameters into consideration, we conclude t h a t the species formed is A2B. A similar analysis for the non-cognate complex formed with t R N A w l gave the same results. Also, as expected, the magnitude of (~l/A)max (at the same constant angle) was the same for both the cognate and non-cognate complexes. The experimental d a t a were further analysed b y comparing t h e m with curves generated for different stability constants, assuming the complex A2B. Figure 2 TABLE 1

Molecular parameters of the complex f o r m ~ between lysine :t R N A ligase and SR N A L~ Complex

Radius of gyration (A) Molecular weight Volume (43 )

Observed

Caloulated~f

Native ligase~

57.5 236,000 600,000

252,600 631,000

37-5 114,000 295,000

tAssumiug the complex A~B. ~0sterberg e$ a/. (1973); included for comparison.

X-RAY

SCATTERING

OF tRNA

LIGASE

COMPLEX

387

E

0.5

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30

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70

50 B (,~M)

I

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150

FIO. 2. E x p e r i m e n t a l data, (A~/`4)/(zJ~T/`4)=== reraua/~ (the t o t a l t R N A "'~" conon), for p H 6-85 w i t h o u t Mg =+, compared w i t h theoretical curves generated for a n A=B complex w i t h log/~s z = 11.4; one curve corresponds to `4 ----24.3 a n d t h e other to -4 = 44.3/zM. T h e e x p e r i m e n t a l p o i n t s correspond to t h e following values of t o t a l concentration of ligase (`4) a n d scattering angle (28): • , ~ = 24.3/zM, 2 8 = 2 . 9 m r a d ; Z3, ,4=24.3/z~r, 2 # = 4 . 9 m r a d ; O , , 4 = 4 4 . 3 / ~ , 2 8 = 2 . 9 m r a d ; O , , 4 = 4 4 . 3 / ~ , 28 ----4.9 mrad.

shows the data obtained at pH 6.85 in the position of best fit relative to the calculated curates.

For complexes of lysine:tRNA ligase with its substrata tRNA, the stability constants decrease successively with increasing pH in the range investigated (Fig. 3). On the other hand, the stability of the complex formed with non-substrata tRNA (tRNA v=l) is virtually independent of pH in this range. Consequently, at low pH, the complex with substrata tRNA is much stronger than that with non-substrate tRNA (cf. Fig. 3). For instance, at pH 6.85, the ligase binds tRNA r'y8 more than ten times stronger than tRNA TM.

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FzG. 3. The stability constants (log/~21) of t h e complexes between lysine: t R N A lJgase (yeast) a n d two t R N A molecules, t R N A TM a n d t R N A TM, as a function of p H . The experimental points correspond to t h e following Mg 2+ concentrations: 10 mM-Mg 2+ ( V , V ) , 1 mM-Mg s+ (Z~, A), 0.1 m ~ - M g 2+ ( O , 0 ) , a n d 0 m~r-Mg 2+ (f-J, II), where t h e open symbols s t a n d for t R N A "'yB a n d t h e filled symbols for t R N A TM. The f~JZ.drsu~ curves, for t h e t R N A Ly~ complex, are calculated using e q u a t i o n (A14) in t h e Appendix. As indicated b y t h e daa/=e~ lines t h e stability constants for t h e t R N A TM complex are virtually i n d e p e n d e n t of p H .

388

R. 0 S T E R B E R G E T AL.

The general shape of the curve relating the stability of a certain complex to pH is the same both with and without Mg2÷ (Fig. 3). However, in the presence of Mg2÷ there is a marked decrease in stability for the whole pH range investigated. For instance, log f12~for the tRNA ''ys complex at pH 6.8 and 1 mM-Mg2+ decreases 1.9 logarithmic units compared with the stability constant in the absence of Mg2÷. The stability of the complex with non-substrate tRNA (tRNA TM) also shows a similar decrease in the presence of M_g2+. Increasing the Mg2+ concentration from 1 mM to 10 m~ makes essentially no difference to the stability. Lowering the Mg2+ concentration to 0-1 m~ gives a stability constant intermediate between that of the complex without Mg2+ and that obtained for the complex at > 1 m~-Mg ~+ (Fig. 3). (b) Moleoular parameters In order to calculate the molecular parameters of the ligase:tRNA complexes the scattering curves must first be corrected for the contributions of free ligase and free tRNA. This correction is small when the stability of the complex is high. For the calculation of the molecular parameters we have consequently used data from experiments at low pH (pH 6.85) and without Mg2+, where a high stability is obtained. As indicated by the apparent radius of gyration, ~, the same complex was formed in each experiment. Thus, within the experimental error, i~ was found to be the same when the data were analysed from at least one solution of each series, i.e. one solution for each pH and Mg2+ concentration including both cognate and non-cognate systems. In order to correct the scattering curve for the intensity contribution of the free molecules, their concentrations were first determined from the stability constant. Then the absolute intensity for each free concentration was estimated by interpolation from a series of small-angle scattering curves, which were recorded separately for both the ligase and the tRNA. After correction, the resulting data were smoothed using a spline function that m~nlmizes the second derivative of the curve (Reinsch, 1967) (of. Schelten & Hossfeld, 1971). The resulting smoothed data werethen corrected for the length co111mation effect using the computer program described by Heine (1963). The desmeared data from which the molecular parameters were finally calculated were obtained for two different concentrations of ligase and tRNA: (1) A = 44 p~, B ---- 76 pM and (2) A = 2 4 / ~ , B = 38/~M. The corrections for free molecules were 10.8 and 12-8~/o of the zero angle intensities.

(i) Radi~ of gyration When the logarithm, of the desme~red intensities, corrected for free molecules, were plotted against (28)2, the data at low values of 20, 0.0029 to 0-0085 radian, could be fitted to straight lines. This gave, within experimental error, the same radius of gyration (57.5-t- 1-5 A) for the two concentrations used (Table 1). The value determined before slit correction and before correction for free molecules was 53 A. (ii) Molecular weight The molecular weight of the ligase complex with tRNA Ly8 was estimated to be M = 236,000 (Table 1), using the following equation X x a 2 x/o/Po M = (z~ _ ~ 2 ) 2 x d x o'

(2)

X-RAY

SCATTERING

OF CRNA LIGASE

COMPLEX

389

where I0]P0 is the absolute scattered intensity, ~1 the partial specific volume, K a constant (K -~ 21.0), "a" the distance between the sample and the plane of registration (20-5 era), d the thickness of the sample (0-0943 era), c the concentration of the sample in g/ml, zl the number of moles of electrons per gram of dissolved substances, and ~ the electron density of the solvent (Kratky, 1963). The quantity/0]Po is the ratio between 1o, the slit corrected intensity at zero angle, and Po, the intensity of the primary beam. The partial specific volume used for the complex, ~i ~ 0.715, is the weight average of the partial specific volumes for the ligase, ~1 ---- 0-734 (Rymo e$ a/., 1972; ~}sterberg e~ al., 1973), and for the tRNA, ~ ----0.54 (Pilz e~ a/., 1970c). The zl value was the weight average of the zl values for the protein, 0.535 (Pi]z et al., 1970a), and for the tRNA, 0.572 (calculated from the primary structure of a tRNA T'ys molecule (Smith et al., 1971)). The value of M was determined by extrapolating linearly to zero concentration the values (226,500 and 230,700) obtained for the two different concentrations used. A molecular weight of 252,600 was calculated for the complex A2B, using a molecular weight of 114,000 for the ligase (0sterberg et al., 1973) and 24,600 for the tRNA (Smith et al., 1971). The difference between the calculated value, M -~ 252,600, and that derived from equation (2), M----236,000, is only 7~/o, which is within the experimental error.

(iii) Volume The volume, V, was determined to be 600,000 _~3 (Table 1), using the following equation (Pored, 1951) V = 0"582aalo/(~. (3) Here, 0.582 is a constant corresponding to the CuK~ wavelength, h ~ 1.542 A, and is Porod's invariant, which is defined by the equation

The quantities (~ and I' have not been corrected for the collimation effect; m is the distance in cm between the detector slit and the primary beam in the plane of registration; I ' is the normalized intensity, lilt, where c is the concentration of the complex (g/m]). For ~ > 0.65 cm the scattering curve followed a course proportional to 1/~ 8. Due to the uncertalnty in the assumption of a uniform electron density, the value obtained for the volume of the complex should only be considered as a first appro~mation. It is, however, in good agreement with the value calculated for the complex A2B (631,000 A 3) using a volume of 295,000 A 3 for the ligase (~sterberg et al., 1973) and 41,500 A 3 for tRNA (Pflzet al., 1970a) and assuming that there is no contraction or overlapping.

(iv) Shape of the complex The experimental scattering curve was compared with theoretical curves calculated for triaxial bodies using the procedure described by Pilz et al. (19705). Assuming that the shape of the ligase molecules in the complex is the same as that determined previously for the native enzyme (Osterberg et a/., 1973), i.e. an ellipsoid with the semi-axes a----62.7, b = 50.1, and c----23.5 A, we have tried to determine the orientation in space of the two ligase molecules and the tRNA molecule in the complex A=B.

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As a first appro~mation, the tRNA molecule was introduced in the form of two separate bodies. We chose two ellipsoids of rotation having the following semi-axes: a = 38.5 A, b ---- c = 13.0 A; and a ---- 20-0 A, b = o -----13.0 A. These two ellipsoids, which to some extent penetrate into each other, form an angle of 90 ° between their major axes (Fig. 4). This tRNA model, which is only a rough approximation based on the X-ray crystallographic data reported for tRNA T M by Suddath ~ al. (1974), (cf. K~m ~ al., 1974), gives the same radius of gyration (25 A) and volume (41,400 A 3) as has previously been reported for tRNA (see e.g. Lake & Beeman, 1968; Ninio ~., 1969,1972; Pllz ~ ,J., 1970a). When the experimental data were compared with the theoretical curves calculated for this tRNA model combined with the ligase molecule model (0sterberg ~ al., 1973), it was first found that a "sandwich" complex (where the tRNA model is situated between the two ligase ellipsoids with their a, b and c semi-axes parallel) gave a radius of gyration of only 49 A compared with the observed value of 57.5 A. Hence such a

-0-5

-I-0 b

i

-Z.5

o, -2.0

-2.5

-3.0 -2.0

-I-5

-J~O

Log s

FIG. 4. Experimentally observed scattering curve from the complex of lysine : t R N A ligase and t R N A L~" from yeast (. ) compared with theoretical scattering curves calculated for 2 models involving $ major ellipsoids representing the ligase molecules and 2 m l , o r ellipsoids (tRNA). The u-axes of ~he ligase ellipsoids form a right angle between each other, and the t R N A ellipsoids situated within this angle, interact with the enzyme ellipsoids. The a-axes of all 4 ellipsoids are in the same plane with the b-axes parallel. (a) About 50% of the t R N A ellipsoid volume is within the enzyme ellipsoid volume; (b) the t R N A ellipsoids barely touch the enzyme ellipsoids. Both models give a radius of gyration of 57.5 A; o = (4~/A)sin O, where A is the wavelength used, A = 1.54 A, and 0 the half scattering angle. For further details, see the text.

X - R A Y S C A T T E R I N G OF t R N A L I G A S E C O M P L E X

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model cannot explain the experimental data. However, if the two ligase ellipsoids were arranged with their a semi.axes at right angles to each other and their b semiaxes parallel and with the tRNA in the angle between the enzyme molecules, as shown in Figure 4, the radius of gyration calculated for the model (57-5 A) agreed closely with that observed. As can be seen in Figure 4, this model gives the best fit between the theoretical and experimental curves, if the tRNA molecule is assumed to be partly engulfed by the two protein ellipsoids (Fig. 4(a)).

4. D i s c u s s i o n

The results presented in the previous sections are consistent with the formation of a complex between lysine:tRNA ligase and tRNA, containing two ligase molecules and one molecule of tRNA. The existence of a species, A2B, is supported both by the analysis of the equilibrium data, which shows a close agreement between the experimental data and the theoretical curve generated for this complex (Fig. 2), and the molecular parameters, which gave a molecular weight of 236,000 and a volume of 600,000 A 8 for the complex, in good agreement with the molecular weight (252,600) and the volume (631,000) calculated for the species A2B. The lysine :t RNA ligase forms similar complexes both with its substrate, tRNA Lyre, and with the non-substrate tRNA TM. However, while the stability of the complex with non-substrate tRNA is virtually independent of pH, there is a marked increase in the stability of the complex with substrate tRNA at low pH (Fig. 3). A similar dependence of the stability on pH has been noted for several other complexes of ligases and tt~eir cognate tRNAs (Mitra et al., 1970; Hdl~ne et al., 1971 ; Schoemaker & Seblmmel, 1974). The presence of Mg2+ decreases the stability of the complex both with tRNA Lys and tRNA TM. The pH dependence of the stability constants (cf. Fig. 3) is analysed and discussed in the Appendix. The results suggest that lysine:tRNA ligase interacts with its tRNA substrate via two different kinds of acid-base groups, one involving about seven protons (pK -~ 4.8) and the other involving about two protons (pK ~ 6.8). The magnitude of the free energy change (--3.2 kcaI/mol) indicates the formation of hydrogen bonds. It should, perhaps, be pointed out that the formation of hydrogen bonds v ~ carboxylate groups (pK = 4 to 5) might facilitate the release of the amlnoaeyl-tRNA product from the enzyme. The breakage of these bonds, which form at neutral pH, could result from a slight alkaline pH change, leading to the dissociation of protons and the unmasking of negatively charged carboxylate groups which would repel the tRNA molecule. The analysis of the shape of the complex, comparing experimental data with data generated for a set of triaxial bodies, indicates that our results can be explained by the model shown in Figure 4(a). Here, we assume that the ligase molecules in the complex can be described by two ellipsoids which interact with their a semi-axes at right angles. The tRNA model consists of two ellipsoids which are arranged in the form of an L (Suddath et al., 1974; Kim et at., 1974) and this L-shaped tRNA model is supposed to interact with the enzyme ellipsoids. The best agreement with the experimental data is obtained when the tRNA ellipsoids are partly engulfed by the protein ellipsoids (Fig. 4(a)). This model has the same radius of gyration as that observed, 57.5 ~. Assuming that the two Iigase molecules interact at right angles (Fig. 4(a)) a large area of contact between the supposedly L-shaped tRNA and the

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R.

0STERBERG E T A L .

enzyme is obtained, consistent with the observation that a ligase protects bound tRNA against nuclease (Lagerkvist & Rymo, 1969; H6rz & Zachau, 1973). Although the evidence for the existence of a complex of the type A2B between the lysine enzyme and tRNA is very convincing, we cannot yet decide whether the formation of such a complex is a peculiarity of this particular enzyme or whether it represents a mechanism of more general importance. Since the native lysine enzyme is of the ~2-type with two identical subunits (Rymo el al., 1972) the enzyme moiety of the complex A2B will have the subunit composition ~4. Looking at the subunit composition of the known ligases, one is struck by the fact that some of them, even as native enzymes in the absence of tRNA, are large enough to be comparable to the ~4-form of the lysine enzyme. The phenylalanine ligases from Escheri~hia coli (Hanke al., 1974) and yeast (Fasiolo ~ al., 1974) both have the subunit composition ~2fl2 with molecular weights of 267,000 and 260,000, respectively. The methionine ligase from E. cell also presents a similar situation in as much as it contains two subunits where each subunit is made up of two identical or at least very similar domains giving the native enzyme a molecular weight of 180,000 (Fayat & Wailer, 1974 and references therein). One might then ask why these enzymes are so large compared with the average native ligase which has a molecular weight around 100,000 or, in some cases, even less. It seems reasonable to assume that being large could be a distinct advantage for an enzyme which must simultaneously recognize several structural features of a macromolecular substrate where the structures recognized might well be situated far apart on the surface of the substrate. I t is then tempting to speculate that perhaps all ligases, in order to recognize their tRNA substrates, must aggregate to a minimum molecular weight, and that the lysine ligase from yeast, when it forms a complex with tRNA which contains the enzyme as an a,-structure, is only conforming to a pattern which is common to all ligases. The phenylalanine and methionine enzymes would then be variations of this more general scheme in the sense that they retain their highly aggregated structure even as native enzymes. In order to elucidate these questions we are presently investigating the valine ligase from yeast, which contains only one peptide chain per molecule of native enzyme where the chain is made up of two identical or very similar domains, and the lysine ligase from E. cell, which is markedly different from its yeast counterpart in terms of molecular weight and amino acid composition (Rymo et al., 1972; Bruton, 1975). Preliminary results indicate that these enzymes also form dimers when they bind tRNA. We would like to express our sincere thanks to Professor Dr O. Kratky, Institut ffir Physikalische Chemic der Universit~t, Graz, for the Lupolen sample andto Mr R. Ligaarden for technical assistance. This work was supported by a grant from the Swedish Natural Science Research Council to one of us (R. 0.) and from the Swedish Cancer Society to a second author (U. L.). REFERENCES Bruten, C. J. (1975). Bioc~era. J. 147, 191-192. Chirikjian, J. G., Wright, H. T. & Fresco, J. R. (1972). Pro~. Nat. Acad. Sc£, U.S.A. 69, 1638-1641. Fasiolo, F., Remy, P., Pouyet, J. & Ebel, J..P. (1974). Eur. J. Biocl~m. 59, 227-236. Fayat, G. & Waller, J.-P. (1974). Eur. J. Biovhem. 44, 335-342. Gillam, I., Blew, D., Warrington, R. C., yon Tigerstrom, M. & Tener, G. M. (1968). Biocl~mis~ry, 7, 3459-3468.

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